JP2005533041A - Sterilization, stabilization and storage of functional biological materials - Google Patents

Abstract

Disclosed is the sterilization, stabilization and storage of functional biological materials. Disclosed are methods for sterilizing functional biological materials, biochemical entities and biologically active molecules and storing those materials at room temperature. Sterilized and stabilized products according to the present invention significantly reduce or eliminate biological contaminants in the titer, while maintaining their overall function. These materials may have mainly a structural function such as bone, or may have an active molecular function as in the case of hemoglobin or an antibody. Treatments that stabilize sterilized biological materials include material temperature changes, radiation dose control, irradiation time optimization, oxygen content control, use of stabilizers, pH specifications, inclusion of specific solvents, And changes in the type of radiation used. Sterilized and stabilized biological products so treated can be stored at room temperature and are much more useful than traditional lyophilized or cryopreserved biological materials And easy to use.

Description

This application claims the benefit of US Provisional Application No. 60 / 387,177, filed Jun. 7, 2002, which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION The present invention relates generally to the fields of biochemistry and medicine, and more particularly to functional biological materials, biochemical (presence) entities, and biologically active molecules such as Blood components, proteins, other cytoplasmic components, intact human and animal tissues, and sensitive and desensitized, including hemoglobin (in erythrocytes and independent), antibodies, cytokines, both forming and non-forming components , And storage by irradiation of materials that act as antigens for testing vaccines.

  Biological materials have been used for many years in a wide range of applications ranging from human and veterinary applications, diagnostic applications, and experimental or chemical processes. Because these materials are often biologically active, that is, they can perform the same or similar structural, enzymatic, or other molecular functions as performed in the original organism or plant. Can be used as a diagnostic, prophylactic or therapeutic agent. Despite the enormous benefits of biological materials, there are also risks in their use. These materials can be contaminated with a variety of biological contaminants ranging from viruses to bacteria and the like. These contaminants can cause serious health problems when transmitted to humans and animals. These contaminants have the ability to reduce or even destroy the effectiveness of the contaminating material.

  Although there are many techniques for testing and screening biological materials for biological contaminants or pathogens, these screening procedures have drawbacks. These techniques are not always reliable, are expensive, and can only test very specific contaminants. It is much more effective to reduce the risk of extensive contamination. Many techniques, including heat treatment, chemical solvents, and radiation, are used to reduce the risk of contamination. It has been found that heat treatment often adversely affects the biological activity of the product and reduces the efficacy of the product. Chemical solvents, disinfectants, and antibacterial agents are used to reduce the possibility of contamination, especially the opportunity for bacterial growth after packaging and before use. These solvents and drugs can be harmful when in contact with humans and often must be removed from the product prior to use in humans.

  Radiation treatment is another method for sterilizing products. Radiation is extremely effective in reducing a wide range of biological contamination, especially including bacteria and viruses. The literature published in this field shows that the technique of radiation sterilization is very flexible, and those skilled in the art will know the type of irradiation used, oxygen content, pH, irradiation dose, temperature, solvent, stabilizer, etc. Can be manipulated to modify the product's response to radiation. However, most of the work done so far in developing radiation treatment plans has focused on ways to inactivate contaminants with little change in biological material in both structure and biological function. Are combined. Such sterilized, stabilized products are functional, i.e. structurally the same or similar to those performed in the original organism or plant so that they can be used as diagnostic, prophylactic or therapeutic agents. It should be able to perform enzymatic, or other molecular functions.

A. One disadvantage of blood and blood components prior art is the preparation of blood and blood components Medical. Extensive injury and medical procedures require the administration of whole blood or various blood components. Not all patients require whole blood, and indeed the presence of all blood components can cause medical problems. Individual blood fractions can be stored under special conditions that are most suitable to ensure their biological activity at the time of transfusion. For example, when the processing center receives donor blood, red blood cells are separated and stored in various ways. Such cells are packaged in citrate-phosphate-dextrose for up to 5 weeks at 4 ° C., generally having a volume of 200-300 ml and a hematocrit value (expressed as a percentage of red blood cell volume) of about 70-90%. Can be stored as red blood cell units. Red blood cells can be treated with glycerol and then frozen at −30 ° C. to −196 ° C. and stored in glycerol solution for up to 7 years, but must be kept frozen at low temperatures in order to hold well for transfusion. I must. Both of these methods carefully maintain storage temperatures, avoid disruption of the desired biological activity of red blood cells, and are reasonable for actual transfusion use in accordance with the American Association of Blood Bank standards. It is necessary to give 24 hours of survival time to at least 70% of the transfused cells considered to be at the level.

  One known method for storing red blood cells is the freezing (lyophilization) of red blood cells, which allows such cells to be stored at room temperature for extended periods of time and easily reconstituted in mammals. This is because it can be used. When many previous methods lyophilized red blood cells (RBC) in, for example, aqueous or phosphate-buffered saline (PBS) solutions, the reduced cells are unable to metabolize cells and cellular hemoglobin is Damaged to the extent that oxygen cannot be transported. Lyophilized, reduced, glutaraldehyde fixed erythrocytes are mainly used for agglutination assays.

  It is well known that low dose gamma irradiation of blood can be used to prevent graft-versus-host reaction diseases associated with blood transfusions. Graft-versus-host response disease (GVHD) occurs when donor lymphocytes are transplanted into sensitive recipients. These donor lymphocytes proliferate and damage target organs, particularly the bone marrow, skin, liver, and gastrointestinal tract, which can eventually die. The disease was originally thought to be a complication of blood transfusion for recipients of allogeneic bone marrow transplantation to patients who received intrauterine blood transfusion and total body irradiation. GVHD is also found in immunologically ineligible patients exposed to donor lymphocytes by infusion of cytoplasmic blood material, or rarely, by transplanted organs. Finally, the most commonly reported environment for transfusion-related GVHD (TA-GVHD) is the biologically relevant or immunologically ineligible receiving of blood from the same HLA donor It is a person. Products relevant in the case of TA-GVHD include non-irradiated whole blood, packaged red blood cells, platelets, granulocytes and fresh non-frozen plasma. It does not contain frozen deglycerolated red blood cells, fresh frozen plasma and cryoprecipitate.

  Of course, the term “blood product” discussed herein can and usually includes whole blood (and fractions thereof), platelets and / or red blood cells. As used herein, the term “leukocyte” encompasses the general division of leukocytes, including mononuclear cells and neutrophils, lymphocytes, and all other cells found in the blood other than red blood cells and platelets. Shall. In addition, a substantially “cell-free” blood product may contain certain white blood cells. Currently, gamma irradiation of blood products is the only known method for the prevention of GVHD associated with blood transfusions. The most common radiation sources are cobalt-60 and cesium-137. Most blood centers have a nominal dose of 25 Gy to deliver more than 15 Gy to any area of these isotope bags to inactivate lymphocytes in the transcytoplasmic product.

  One of the many problems plaguing the use of blood transfusions is the transmission of the motive that causes the infection. Since pathogenic organisms are found in various fractions of whole blood, the risk of disease after transfusion varies depending on the blood product or component used. Some products prepared for human, veterinary or laboratory use may contain undesirable and potentially dangerous contaminants such as viruses, bacteria, yeasts, molds, mycoplasmas and parasites. Many of these pathogens are clinically very important because they are not only dangerous for recipients, but also dangerous for physicians and other hospital personnel who handle blood and blood products It is. It is therefore very important to ensure that such products are not contaminated before use. This is especially critical when the product is administered directly to the patient, for example, by blood transfusion, organ transplantation and other forms of human therapy.

B. Biological molecules prior art methods which act as antibodies and antigens, antibodies, and is defective with regard storage of a biological molecule that acts as an antigen. Antibodies are currently widely used in diagnostic tests. In fact, many bacteriological and pathological diagnoses are made by tests that rely on the reaction of antibodies with specific antigens. Such tests include a simple hemagglutination assay used to subject blood to an ELISA (enzyme-linked immunosorbent assay) test. By storing the antibody at room temperature, the antibody can be used immediately in a field hospital, such as a field hospital for military action, so that accurate and rapid diagnosis can be achieved in areas where there is no established research facility. Currently, the need for refrigeration makes it very complex to treat patients with antibody formulations. Accordingly, the production, transportation, and storage of antibodies are prohibitively expensive and often not cost effective.

C. Tissues and organs for use in medical applications Currently, cryopreservation is the primary means for preserving allograft tissue and transplant material derived from tissue. Recently, many infections have originated from bones and tendons stored in banks. Irradiation sterilization can prevent transmission of both bacterial and viral pathogens. Furthermore, storage at room temperature after irradiation greatly simplifies the preparation, storage and use of allograft tissues and their derivatives including bone, collagen and acellular dermis.

  Previously, most procedures included products made from biological materials, biochemical entities, and biologically active molecules with respect to specific contaminants rather than removing contaminants from those products. A method of screening or testing is involved. Products that have been tested positive for certain contaminants are simply not used. Examples of screening procedures include testing for specific viruses in human blood obtained from blood donors. However, such a procedure is not always reliable. This reduces the value or certainty of the test with respect to the conclusions associated with false negative results. In some cases, for example in the case of acquired immunodeficiency syndrome (AIDS), for example when testing for human immunodeficiency virus (HIV), false negative results can be life threatening. Furthermore, in some cases, it may take weeks instead of months to determine if the product is contaminated.

  More recent research has focused on how to remove or inactivate biological materials, biochemical entities, and contaminants in biologically active molecules before using them. It is squeezed. Such methods include heat treatment, filtration, addition of chemical deactivators and treatment with gamma rays or other radiation. There are many disclosures in the literature that gamma irradiation is effective in killing viruses and bacteria. In fact, some researchers conclude that gamma irradiation is most effective in reducing or eliminating virus levels. Biological materials are sterilized by irradiation, but these materials are traditionally stored after processing by refrigeration or freezing. Viral inactivation by severe heat sterilization is unacceptable because it can also destroy the functional components of blood, especially erythrosites (erythrocytes) and plugs (platelets) and sensitive plasma proteins.

  From the above, it effectively removes biological contaminants for sterilizing products containing biological materials, biochemical entities, and biologically active molecules, while There is a need to provide a method that does not adversely affect it. Examples of undesirable biological contaminants include viruses, recently yeast, mold, mycoplasma and parasites. Therefore, before infecting recipients with such pathogens by transfecting pathogenic viruses, microorganisms, or parasites present in human whole blood or blood products with such products into the recipient. A safe and economical way to eradicate, is highly desirable. At the same time, by properly removing blood contamination, the threat of infection to hospital staff who must handle these body fluids is also avoided. This need is even more significant in blood banks that store and process donor blood and blood products. Pathogenic viruses, microorganisms or parasites present in human or animal tissue (eg skin, bone, membranes, and tendons) of biological material before such tissue is introduced or transplanted into the recipient It would also be equally desirable to provide a safe and economical method and apparatus that eradicates and thereby prevents infection of recipients due to such diseases. Thus, the present invention addresses these and other needs.

SUMMARY OF THE INVENTION Briefly and generally, the present invention relates to the preservation of biological materials, functional biochemical entities and biologically active molecules by irradiation. The present invention requires the need to freeze or refrigerate biological materials, biochemical entities, products derived from biologically active molecules, at ambient or room temperature, in particular lyophilization before irradiation or after irradiation. Without further preparation methods for storage. More particularly, the present invention relates to antibodies, peripheral blood cells (eg, red blood cells and platelets), plasma protein fractions (eg, albumin and coagulation factors) collected from whole blood (eg, blood of a patient infected with a virus), body fluids (urine) Including, but not limited to, cerebrospinal fluid, amniotic fluid, and synovial fluid), ex vivo media used in the manufacture of antiviral vaccines, and cell culture media (eg, fetal bovine serum and bovine serum). Potential biological contaminants (e.g., viruses, bacteria, yeasts, molds) of the composition, or products derived from such compositions, and solutions of sugar, amino acids, peptides, and lipids for intravenous nutrition. , Mycoplasma and parasites) inactivation. The invention further encompasses monoclonal antibodies, botulinum toxins and plant-derived proteins, hemoglobin (in erythrocytes and independent), antibodies and vaccines, but not limited to blood system proteins and biologically Relates to the derived protein. It is not a prerequisite for the value of the present invention whether the contamination is precedent or potential.

  Most research in radiation treatment methods to date has focused on methods to inactivate contaminants with little change in biological material in both structure and biological function. The present invention is based on known radiation treatment methods, where the radiation treatment is not only sterilized, but without the apparent control of the temperature of the sterilized product following irradiation, the biological material, the biochemical reality. It is disclosed that it can also be effective in preserving the function of products and biologically active molecules. With the unique storage and processing methods of the present invention, sterilized products can be stored at room temperature and maintain efficacy with respect to the biological activity and mechanism of action of the product.

  This is a significant advance for a number of reasons. Most importantly, the ability to store the treated product at ambient temperature reduces the risk of compromising the effectiveness of the material without strict adherence to handling procedures. In many parts of the world, even refrigeration is a luxury. This has a dramatic impact on the health of the population in these areas, as medicines and vaccines are routinely inactivated because they cannot be refrigerated. The ability to supply vaccines and biological materials that are stable at ambient temperatures in the developing world will have a major impact on the health of people in these regions. Storage at ambient temperature saves storage and transportation costs, and in many cases provides a more convenient, easy-to-use product and has a significant impact in developed countries by producing it at a low cost. Let's go.

  One opportunity to use the present invention is the preparation of medical blood and blood components. The present invention relates to whole blood samples, or biologically derived proteins or structures (plants or animals) so that the risk of transmission of infectious diseases, in particular viral diseases, is substantially reduced or eliminated. Sterilized and stored (including cells and tissues, such as cells and tissues). The present invention further encompasses a method for producing biological materials that is inexpensive and can be readily supplied to the majority of the medical community. By such methods, biological material can be stored without the need for refrigeration or other processing that can lead to significant cost increases. Furthermore, the present invention provides for the sterilization of biological proteins and the ability to safely store biological proteins at room temperature, which can then be used with greatly reduced risk of contamination by bacteria and special viruses. Stabilization, eg, sterilization of blood antibodies and other chemical components, is disclosed.

  Many functional biological materials can be produced by the method of the present invention. Such a sterilized product can be used in a method for preventing or treating a physical condition or disease because the biological material can be stored at ambient temperature prior to administering an effective amount of the biological material to a patient. Similarly, sterilized and stabilized products formed from irradiated biological materials, biochemical entities, and biologically active molecules can be incorporated into diagnostic test methods and kits, and industrial And can be used as an element in chemical manufacturing. Similarly, nutritional solutions containing sugars, amino acids, peptides and lipids can be sterilized by this method and stored at room temperature (ambient temperature).

  The method of the present invention irradiates one or more biological materials, biochemical (presence) entities and biologically active molecules, preserves their function, and is sterilized and stabilized Includes methods that allow the product to be stored at or near ambient temperature. Functional biological materials suitable for use in the present invention include blood or blood components such as red blood cells, white blood cells including monocytes, immunoglobulins including platelets, clotting factors, mono and polyimmunoglobulins. However, it is not limited to these. Similarly, suitable functional biological materials include animal tissues (including mammals and other animal phylums) such as cartilage, bone marrow (including bone marrow cell suspension), whole or treated ligaments, Tendons, nerves, bones (including demineralized bone matrix), grafts, joints, femurs, femoral heads, teeth, skin grafts, heart valves, corneas, arteries, veins, lipids, Carbohydrates, collagen (natural, afibrillar, atelomeric, soluble, insoluble, recombinant and transgenic, including both native and denatured) and organs (transplant organs such as heart, liver , Lungs, kidneys, intestines, pancreas, limbs, and fingers).

  Similarly, the invention relates to non-cellular materials such as proteins (including recombinant and transgenic proteins), proteinaceous materials, amino acids, peptides, sugars, lipids, enzymes (digestive enzymes such as trypsin, chymotrypsin, alpha-glucosidase and (including iduronodate-2-sulfatase), antigen, marrow, chitin and derivatives thereof (including NO-carboxychitosan-NOCC). The method of sterilization and stabilization of the method of the present invention and the resulting product allows for the measurement of biological materials, biochemical entities and biologically active molecules, the risk of viral and bacterial contamination. And irradiation with a calculated amount of radiation to be effective in sterilizing and stabilizing the biological material.

  In the present invention, the irradiation step can be performed at various temperatures including (but not limited to) a general range from room temperature to a cryogenic temperature including the temperature of liquid nitrogen. In rare cases, it may be advantageous to heat the target sample simultaneously.

  Other features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, illustrating by way of example the features of the invention.

Detailed Description of the Preferred Embodiments The present invention sterilizes and preserves biological materials, biochemical entities and biologically active molecules (biological materials), and one or more potentials therein. Biological contaminants or pathogens such as viruses, bacteria (including intercellular and intracellular bacteria such as mycoplasma, ureaplasma, nanobacteria, chlamydia, rickettsia), yeast, mold, fungi, mononuclear or polynuclear parasitism It relates to a method for reducing the level of worms and / or prions or similar substances that cause infectious spongiform brain disease (TSE) alone or in combination. Current storage methods for such biological materials typically lyophilize the biological material prior to sterilization and / or refrigerate the product after sterilization prior to infusion or use on the human body. . Certain methods previously disclosed use ionizing radiation, but such methods use additional additives and solutions in conjunction with the radiation. For example, some studies have focused on eliminating white blood cells, typically using low doses that cannot effectively inactivate viruses in cells and plasma. The prior art document also does not describe the ability to store red blood cells at room temperature following irradiation. Our method does not require these additional solutions. Numerous studies have shown that red blood cells are not destroyed by the doses required to inactivate known pathogenic viruses. Thus, the present invention is a simple method for storing and sterilizing blood prior to transfusion and is a safe method for recipients.

  The present invention provides a simpler method for storing and storing red blood cells while improving the safety of the material after irradiation. The prior art does not describe storing red blood cells at ambient temperature (eg, room temperature) after irradiation. The prior art also incorporates additional solutions during the irradiation process. It may be beneficial to add a solution that counteracts the potassium leaking from the red blood cells, eg, a buffer that contains or does not contain an agent that binds potassium. This solution may also contain additional glucose in the blood bag to provide an additional energy source for the red blood cells. By simply irradiating red blood cells in a suitable solution environment in their basic storage bag, many known pathogens can be removed and red blood cells can be stored at room temperature.

I. Contaminants As used herein, the term “biological contaminants or pathogens” may adversely affect their recipients by direct or indirect contact with biological materials. Means a contaminant or pathogen (alone or in combination). Such biological contaminants or pathogens include various viruses, bacteria (including intercellular and intracellular bacteria such as mycoplasma, ureaplasma, nanobacteria, chlamydia, rickettsia), yeast, mold, fungi Found in or infecting mononuclear or polynuclear parasites, prions, substances that cause TSE, and other biological materials, biochemical entities and biologically active molecules Substances known to those skilled in the art are included. Other examples of biological contaminants or pathogens include viruses (eg, human immunodeficiency virus and other retroviruses), herpes virus, filo virus, circo virus, paramyxovirus, cytomegalo Virus, hepatitis virus (including hepatitis A, B, and C and other variants), poxvirus, togavirus, Epstein-Barr virus and parvovirus, bacteria (mycoplasma, ureaplasma, nanobacteria, chlamydia, rickettsia) Including, for example, Escherichia, Bacillus, Campylobacter, Streptococcus and Staphylococcus, parasites, eg Trypanosoma and malaria parasites including Plasmodium species, yeast, mold and prions, or similar substances that cause TSE alone or in combination For example, scrapie, kuru, BSE Spongiform encephalopathy), CJD (Creutzfeldt - Jakob disease), Gerstmann-Straeussler-Scheinkler syndrome, and fatal familial insomnia, including without being limited thereto. As used herein, the term “active biological contaminant or pathogen” can be used alone or with other factors, such as a second biological contaminant or pathogen, or a natural protein (wild type or mutant) or antibody, In combination with a biological contaminant and / or pathogen that can cause adverse effects in biological materials and / or their recipients.

II. Biological material Biological material As used herein, the term “biological material” means any substance derived from or derived from an organism. Representative examples of biological materials include cells, tissues, blood, blood components, proteins (including recombinant proteins, transgenic proteins and proteinaceous materials), amino acids, peptides (all natural and synthetic peptides) ), Sugars, lipids, enzymes including digestive enzymes (eg, trypsin, chymotrypsin, alpha-glucosidase and iduronodate-2-sulfatase), immunoglobulins (including monoglobulins and polyimmunoglobulins), botanicals and foods However, it is not limited to these. Preferred examples of biological materials include ligaments, tendons, nerves, bones (including demineralized bone matrix, grafts, joints, femur and femoral head), bone marrow (whole or processed bone marrow cells) Suspensions), teeth, skin grafts, heart valves, cartilage, corneas, arteries, veins, organs (including organs for transplants such as heart, liver, lung, kidney, intestine, pancreas), limbs, fingers , Lipids, carbohydrates, collagen (natural, non-fibrillar, stunted, soluble and insoluble, including both recombinant and transgenic, both native and denatured), enzymes, chitin, and derivatives thereof (NO-carboxychitosan " NOCC ”), stem cells, islets of Langerhans and other transplant cells (including genetically altered cells), red blood cells, white blood cells (including monocytes) and platelets. The present invention is not limited to these.

1. Blood has been previously required but is not available to sterilize blood for transfusion, blood components and blood products, but the function of the formed elements and proteins of the transfusion is quick and safe to preserve Means. Radiation not only provides a means to produce transfusion components with increased safety, but also allows blood components and whole blood previously frozen or refrigerated to be stored at room temperature. Such products and their manufacturing methods provide great benefits because they can be stored at room temperature, making the supply of blood and blood components safer and more readily available. The shelf life of irradiated blood and blood products is also expected to far exceed that of refrigerated blood. Currently, about 20% of transfusion units are stale before transfusion. Due to the increased shelf life, these units that are now obsolete can be used. Therefore, this shelf life extension has the effect of increasing blood supply, ie another beneficial effect of introducing this new technology.

  The present invention relates to the treatment of tissue to remove biological contaminants, and more particularly to the sterilization and storage of blood and blood components. Moreover, the present invention relates to storing red blood cells at room temperature following irradiation. The present invention allows blood bank blood to be transported and stored at room temperature. As used herein, the term “blood component” means one or more components that can be separated from whole blood, including cytoplasmic blood components (eg, red blood cells, white blood cells, and platelets), blood proteins (eg, blood clotting factors, enzymes, albumin, plus Including, but not limited to, minogens, fibrinogen, and immunoglobulins) and liquid blood components (eg, plasma, plasma protein fraction “PPF”, cryoprecipitate, plasma fraction, and plasma-containing composition) is not. As used herein, the term “liquid blood component” refers to one or more liquid, non-cytoplasmic components of whole blood, such as plasma (liquid or non-cytoplasmic portion of human or animal whole blood, found before clotting) and serum (human Or the fluid, non-cytoplasmic part) of whole animal blood after clotting.

As used herein, the term “cytoplasmic blood component” means one or more components of whole blood comprising cells, such as red blood cells, white blood cells, stem cells, and platelets. Survival can be red blood cells, ATP synthesis capacity, cell _{morphology, p 50} value, oxyhemoglobin, methemoglobin and Hemikuromu value, MCV, MCH, and MCHC values, characterized by cellular enzymatic activity, and one or more in vivo survival. Thus, cells that have been lyophilized and then reduced and inactivated with respect to the virus are transfused if the cells are damaged to the extent that they cannot metabolize or synthesize ATP, or the cell circulation is impaired. Usefulness in medicine is impaired. Conversely, irradiated erythrocytes can still perform biochemical functions without the need for nucleic acid synthesis activity. Unlike most other mammalian cells, red blood cells are unique in that they do not have a nucleus and are therefore much smaller than the cell's DNA, and are therefore less susceptible to inactivation by the incident radiation of sterile radiation. Representing a more resistant target, protein elements can still function after irradiation.

  As used herein, the term “blood protein” refers to one or more proteins normally found in whole blood. Representative examples of blood proteins found in animals include both vitamin K-dependent (eg, Factor VII and Factor IX) and non-vitamin K-dependent (eg, Factor VIII and von Willebrands factor) coagulation proteins, albumin, Lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), complement protein globulins (immunoglobulins IgA, IgM, IgG and IgE), but are not limited to these. . Preferred groups of blood proteins include factor I (fibrinogen), factor II (prothrombin), factor III (tissue factor), factor V (proacrelin), factor VI (accelerin), factor VII (proconvertin, serum prothrombin) Conversion), factor VIII (antihemophilic factor A), factor IX (antihemophilic factor B), factor X (Stuart-Prower factor), factor XI (plasma thromboplastin precursor), factor XII (Hageman factor), Factor XIII (protransglutamidase), von Willebrands factor (vWF), factor Ia, factor IIa, factor IIIa, factor Va, factor VIa, factor VIIa, factor VIIIa, factor IXa, factor Xa, factor XIa, factor XIIa, and Factor XIIIa is mentioned. Another preferred group of blood proteins includes proteins found inside red blood cells (eg, hemoglobin), various growth factors, and derivatives of such proteins.

  Plasma and serum (liquid phase after clot formation) are components of blood. Numerous proteins and other factors exist and are useful for a wide range of medical applications. Antibodies are present in plasma and serum. The antibodies can be used for many applications in patient treatment (as in the case of gamma globulin, mixed antibody) and research. The method of storing red blood cells described in detail herein also applies to the production of antibodies and other proteins in that they can be irradiated with such isolates to be sterilized and stored at room temperature. This is very useful for medical fields and laboratory studies. This technique can be applied to antibodies produced from tissue blood obtained from human blood or from the blood of other animals or from the medium in which the antibody is produced. This technology can also be used to produce sterile viral vaccines for storage at room temperature, which greatly simplifies vaccine distribution and administration. In addition, red blood cells can be processed and stored at room temperature and used for hemagglutination blood group testing.

2. Suitable materials for manufacturing bags and other containers for blood bags sterilized and stabilized biological materials, biochemical entities and biologically active molecules include silicones, plastics, and Although foil is mentioned, it is not limited to these. For example, oxygen permeable silicone bags that can be crushed are suitable for storing irradiated biological materials, such as sterilized and stabilized blood products. The use of such bags may be important in converting methemoglobin formed upon irradiation to oxyhemoglobin prior to transfusion. A high oxygen concentration environment may also help to irradiate more effectively with a lower total radiation dose. A flexible, collapsible bag made of poly (ethylene-vinyl acetate) (EVA) plastic is commercially available from the Fenwal Division of Baxter Travenol Laboratories, Inc., Deerfield, Illinois.

  Red blood cells are often washed to reduce the number of white blood cells in the blood. White blood cells can elicit an immune response from the recipient. Due to the risk of contamination with blood bacteria, these washing steps reduce the shelf life of red blood cells to 24 hours. By resealing and irradiating the blood storage bag, the shelf life is greatly extended. The current standard for shelf life following irradiation is 28 days from the time of irradiation or the original expiration date on the unit, whichever comes first. This greatly extends the shelf life of the washed red blood cells.

B. The present invention relates to functional biochemical (presence) realities and biologically active molecules such as hemoglobin (in erythrocytes and independent), antibodies, peptides (natural and synthetic) upon irradiation. Both), vaccines and other antigens, including but not limited to. More particularly, the present invention relates to antibodies, peripheral blood cells (eg, red blood cells and platelets), plasma protein fractions (eg, albumin and clotting factors) collected from whole blood (eg, blood of a virus infected person), body fluids (urine) , Cerebrospinal fluid, amniotic fluid, and synovial fluid), ex vivo media used in the manufacture of antiviral vaccines, and cell culture media (eg, fetal bovine serum and bovine serum), or derived from such compositions The inactivation of potential biological contaminants of the product, such as viruses, bacteria, yeast, mold, mycoplasma and parasites. The present invention further includes a method for producing a whole blood product for storage at room temperature. The present invention further relates to blood system proteins and biologically derived proteins, including but not limited to botulinum toxins and plant derived proteins.

  In another embodiment of the invention, antibodies, clotting factors, growth factors, and other biologically derived proteins, including whole viruses or parts thereof, are described above with respect to blood and blood components. Can be stored with irradiation technology. High dose gamma irradiation inactivates bacteria and viruses and allows the irradiated material to be stored at room temperature. This makes very important biologicals such as polio vaccines that currently have to be refrigerated (as needed, such as in Central Africa where there are few facilities with refrigerators that can store vaccines that require cold storage. Useful in some areas) will be more useful.

C. Biologically active molecule As used herein, the term “proteinaceous material” means any material derived from or derived from an organism comprising at least one protein or peptide. Proteinaceous materials can be naturally occurring materials (in their natural state or processed / purified and / or derivatized), either artificially produced materials (chemically synthesized or recombinant / transgenic techniques). Manufactured and optionally processed / purified and / or derivatized). Representative examples of proteinaceous materials include cell cultures, proteins and peptides produced from milk and other dairy products, ascites, hormones, growth factors, materials extracted or isolated from animal tissue or plant material (insulin ), Plasma and plasma protein fractions (including fresh, frozen and lyophilized), fibrinogen and its derivatives (eg fibrin, fibrin I, fibrin II, soluble fibrin, fibrin) Monomer and fibrin sealant products), whole blood, protein C, protein S, alpha-1-antitrypsin (alpha-1 protease inhibitor), butyl-cholinesterase, antiagglutinin, streptokinase, tissue plasminogen activator ( tPA), erythropoietin ( PO), urokinase, NEUPOGEN (filgrastim, granulocyte stimulating factor), anti-thrombin-3, alpha-galactosidase, iduraonate-2-sulfatase, (fetal) bovine serum / horse serum, meat, immunoglobulin (antiserum. Monoclonal antibody , Polyclonal antibodies and genetically engineered or manufactured antibodies), albumin, alpha-globulin, beta-globulin, gamma-globulin, coagulation protein, complement protein, and interferon. It is not a thing.

III. Radiation As used herein, the term “sterilization” refers to the level of at least one active or potentially active biological contaminant or pathogen found in the biological material being treated according to the present invention. It means lowering. The term “radiation” as used herein means radiation that has sufficient energy to sterilize at least some component of the biological material being irradiated. The types of radiation include particles (particles smaller than atoms, eg neutrons, electrons and protons), electromagnetic waves (from various electromagnetic fields, eg radio waves, both visible and monochromatic and polychromatic light, invisible light, red External, ultraviolet radiation, X-rays, gamma rays, and combinations thereof), sound waves and pressure waves, but are not limited thereto. Such radiation is often described as either ionizing radiation (which can form ions in the irradiated material), such as gamma radiation, and non-ionizing radiation, such as visible light. The sources of such radiation vary, and it is generally important to select a particular source of radiation provided that sufficient radiation is provided at the appropriate time and at the appropriate rate to effect sterilization. is not. In practice, gamma rays are usually generated by cobalt or cesium isotopes, whereas UV and X-rays are generated by machines that emit UV and X-rays, respectively. Electrons are often used to sterilize materials in a method called “e-ray” irradiation where electrons are generated by a machine. Visible light, both monochromatic and polychromatic light, is generated by a machine and can be combined with invisible light generated by the same machine or different machines, such as infrared and UV.

  In this field, by irradiating a biological material sensitive to radiation with a dose effective to sterilize the biological material for a time effective to sterilize the biological material, Methods are known for sterilizing and protecting the biological material from radiation. See US 2003/0012687 A1 (Macphee et al.), 09 / 973,958, now US Pat. No. 6, xxx, xxx, the contents of which are incorporated herein by reference. However, the present invention is based on such prior art disclosures for use as a biological material that can be sterilized, prepared, and stored at ambient temperature for biologically active molecules, such as whole blood samples. The first known use of gamma irradiation is disclosed. It is clear to use safe, effective and inexpensive methods because of the risk of transmission of infectious diseases such as HIV, hepatitis and other viral diseases. The only obvious factors limiting the usefulness of this technology are suitable biologically active molecules (eg blood, blood components, biological proteins, vaccines, viruses and other antigens) and cobalt-60 The availability of a radiation source, or other suitable radiation source. The method of the present invention is low cost and due to the fact that the biologically active molecule does not contain viruses, in particular it does not contain HIV, this method is used for a variety of patients with different needs. It becomes the most attractive means for producing biologically active molecules. The methods of the present invention are often performed at ambient temperatures without the need for cooling, freezing or chemical treatment of the product containing the biologically active molecule prior to performing the method. Some special processing steps in the method can be omitted.

  One of the methods of the present invention provides a prolonged exposure to gamma radiation that substantially reduces damage to products containing biologically active molecules. Typically, the irradiation is performed for 10 hours or more, preferably about 20 to about 40 hours, more preferably about 20 to about 30 hours. The irradiation dose is about 0.5 kGy / hour to about 3.0 kGy / hour, depending on the product to be sterilized and the length of irradiation time. The total dose of irradiation is typically in the range of about 20 to about 32 kGy, which indicates that these levels are effective in reducing the level of contaminants such as viruses. Because it is. High doses of up to 4.0 kGy / min can be irradiated for short periods of up to 5 minutes or longer to sterilize biological products, maintain function, and subsequently store at room temperature.

  Preservation of biologically active molecules (eg whole blood, blood components, biological proteins and viruses) by gamma radiation has many advantages, and such biologically active molecules are currently Possibility to use in unusable areas such as small hospitals, doctor offices, and developing countries around the world. Preparation of irradiated whole blood is inexpensive, simple to perform, and requires only basic materials and a cobalt-60 source. Irradiated whole blood can be stored on a shelf at room temperature and does not require liquid nitrogen or cryopreservation. Irradiated whole blood does not require thawing, washing or rehydration treatments as found in other whole blood preservation methods.

  In one method of the invention, a product containing biologically active molecules can be irradiated, preferably in a form containing less than 20% solids. Thus, certain products can be diluted before irradiation. Processing in a diluted form can also reduce product degradation during irradiation. The choice of diluent depends on the nature of the product to be irradiated. For example, when irradiating blood cells, a physiologically acceptable diluent such as citrate phosphate dextrose will be selected.

  The method of the present invention is useful for treating organic products that are sensitive to radiation. Such products may be susceptible to degradation when irradiated by standard methods. However, irradiation of sensitive products by the method of the present invention is not expected to be harmful to those products. The method is typically applied to biological products, such as, but not limited to, blood and blood components. When irradiating live cells (eg, blood cells), it is advisable to add a scavenger to bind free radicals and other substances that are toxic to the cell. Suitable collectors include, but are not limited to, antioxidants, free radical collectors, and ligands that stabilize the molecule.

  Other aspects of the invention can be practiced by irradiating a sample of a biologically active molecule for a time sufficient to provide a sterilizing dose of radiation. Such doses are therefore calculated using the usual parameters of raw material measurement. Irradiation doses sufficient to effect sterilization are known in the art. Rinsing is not necessary to practice the present invention.

An example of a plan that includes the amount of gamma radiation to irradiate red blood cells and the time to deliver that dose includes the following:
(i) The irradiation dose to be delivered should be 2500 cGy directed at the central part of the container and the minimum dose at other points should be 1500 cGy.
(ii) The time required to deliver the dose should be based on the radiation intensity of the source. The decay of the source should be calculated according to the manufacturer's instructions. The FDA currently recommends that the source be recalibrated annually for cesium-137 and every six months for cobalt-60. The procedure for calculating disintegration included in the operating instructions for the irradiator can be described in the standard operating procedure (SOP).
(iii) SOP should indicate the maximum number of units of blood or blood components that can be irradiated at one time. This is a one-time amount and can be instructed by the manufacturer's procedure based on the company's warranty deadline.
(iv) The total irradiation dose should never exceed 5000 cGy in any part of the container.

  Alternatively, blood can be exposed to radiation doses on the order of 30.0 kGy to kill blood bacteria, viruses, and other potential pathogens, and subsequently store at room temperature.

IV. Examples FIGS. 1-21 summarize data from experiments performed on whole blood using one embodiment of the method of the present invention. Referring now to FIG. 1, blood obtained from a blood bank was irradiated with gamma rays at a total dose of 30 kGy. Blood was collected in USP anticoagulant citrate phosphate dextrose adenine solution (CPDA-1) blood-pack units (Baxter Healthcare Corporation, Deerfield, IL). This blood was stored for 1 week at 4 ° C. after the warranty period and before irradiation. This table lists some characteristics of blood before and after irradiation. It can be seen that pO ₂ , HbO ₂ , and O ₂ saturation are significantly reduced after irradiation. MetHb (methemoglobin), which reflects the oxidation of iron atoms in hemoglobin, is significantly increased. This finding indicates the need to find ways to reduce these changes so that irradiated blood can carry oxygen more easily after transfusion.

  The experimental results demonstrating the effect of whole blood irradiation according to the present invention can be seen in FIG. The design of the experiment that obtained this data is as follows. Whole blood anticoagulated with EDTA in a freshly drawn 5 milliliter (ml) evacuated tube was irradiated by gamma irradiation at room temperature with a dose varying from 0 to 50 kGy in 10 kGy steps. Half of the number of tubes was used for comparison to a pair of tubes supplemented with 0.01 ml of methylene blue 1% solution (10 mg / ml). Prior to analysis, oxygen gas was blown through a tube containing methylene blue for a short time to test whether oxyhemoglobin was formed at a high concentration. After irradiation, blood samples were analyzed at the hospital hematology laboratory using the facility's standard machines. Many results are shown in FIGS.

Referring now to FIG. 2, the experiment demonstrates that hematocrit is maintained after gamma irradiation of whole blood according to the present disclosure. 0.01 ml of a 1% methylene blue solution (10 mg / ml) was added per 5 ml sample tube, equivalent to a pharmacological dose of 1-2 mg / kg used for the treatment of patients with methemoglobinemia. As shown in FIG. 3, which is a partial pressure curve of oxygen after adding methylene blue and exposing to oxygen, the pO ₂ in the sample tube is high. With reference now to FIG. 4, the experimental data demonstrate that the oxygen saturation of hemoglobin in the methylene blue treated and irradiated sample is uniformly high (close to 100%). Saturation at 2 and 3 Mrads for tubes without methylene blue is believed to be due to oxygenation when the blood is agitated in preparation for testing. This demonstrates that oxygen gas can be bound by methylene blue treatment and exposing the irradiated blood to oxygen. The fraction of hemoglobin bound to oxygen decreases with increasing radiation dose, as shown in FIG.

  With reference now to FIG. 6, experiments demonstrate that increasing radiation correlates with increasing methemoglobin content. MetHb levels for tubes without methylene blue are unexpectedly low from other observations, including the observation of FIG. 1 above. Similarly, and as shown in FIG. 7, the fraction of hemolyzed free hemoglobin is much higher in the tubes without added methylene blue. Furthermore, the sodium concentration in the blood sample tube demonstrates relative hyponatremia. The comparative and methylene blue-containing samples show similar values (Figure 8). Similarly, and as shown in FIGS. 9 and 10, the white blood cell count and red blood cell count are not significantly affected by irradiation or the presence of methylene blue.

  Here, with reference to FIG. 11, experimental data show that the hemoglobin content gm% is affected by irradiation. This is believed to reflect artifacts resulting from radiation-induced changes. Other investigators have now reported post-radiation macrocytosis with increased hematocrit, as seen in some samples (FIG. 12). As shown in FIG. 13, the average red blood cell volume appears to increase with irradiation, as other researchers have noticed. 14 and 16 show the mean erythrocyte hemoglobin and mean erythrocyte hemoglobin concentration measured in experiments conducted according to the present invention. These data show that the red blood cell distribution width (FIG. 17) is not dramatically different except for one point that is believed to represent some sort of systematic error. These data also show an increase in platelet count, possibly as a result of blood concentration (FIG. 15), but the mean platelet volume has not changed dramatically under experimental conditions (FIG. 18).

Samples obtained from irradiated units of whole blood were examined by appropriate hematological staining under a microscope, and intact red blood cells were observed after 30.0 kGy gamma irradiation (FIG. 19). Biochemical analysis showed reversible methemoglobinemia with standard techniques used in actual medicine. Addition and oxygen exposure of methylene blue is believed from the Fe ⁺⁺⁺ state iron is oxidized in the hemoglobin, it is effective to convert the Fe ⁺⁺ state normally present in functional hemoglobin. In addition, silicone blood bags can assist in this conversion by diffusing oxygen into the blood and onto Fe ⁺⁺ atoms prior to transfusion.

  In one embodiment of the invention, whole blood obtained from HIV and hepatitis negative donors can be obtained at the time of expiration. Such HIV-infected blood can be stored at 4 ° C. and irradiated with 30 kGy gamma irradiation. The blood can then be shipped and stored for long periods at room temperature.

  FIG. 20 shows the ability of anti-A antibody to agglutinate Group A red blood cells. For this experiment, anti-A antiserum was divided into two aliquots. One was stored in a refrigerator, the other was irradiated with 30 kGy gamma irradiation and stored at room temperature for one month. In this experiment, the ability to aggregate two antiserum group A cells was tested in serial 1: 2 dilutions of antibody in physiological saline in round bottom wells containing group A cells. The antibody titer of the refrigerated antibody was 1:80, while the antibody titer of irradiated and stored at room temperature was 1:40.

  While particular forms of the invention have been illustrated and described, it will be apparent to those skilled in the art that various modifications can be made without departing from the spirit and scope of the invention. More particularly, the present invention is not limited to the storage of specifically cited biological materials, biochemical entities and biologically active molecules, many of which have not been specifically described. Obviously, it can also be applied to the storage of suitable biological materials. Likewise, the invention is not limited to special medical device structures that can perform the disclosed methods and products of the invention. Accordingly, the invention is not limited except as by the appended claims.

FIG. 1 is a table showing characteristics of the test sample. FIG. 2 is a graph showing the relationship between Hct and Mrad with / without methylene blue. FIG. 3 is a graph showing the relationship between pO ₂ and Mrad with / without methylene blue. FIG. 4 is a graph showing the relationship between O ₂ SAT and Mrad with / without methylene blue. FIG. 5 is a graph showing the relationship between FO ₂ Hb and Mrad with / without methylene blue. FIG. 6 is a graph showing the relationship between FMetHb and Mrad with / without methylene blue. FIG. 7 is a graph showing the relationship between FHHb and Mrad with / without methylene blue. FIG. 8 is a graph showing the relationship between Na and Mrad with / without methylene blue. FIG. 9 is a graph showing the relationship between WBC and Mrad with / without methylene blue. FIG. 10 is a graph showing the relationship between RBC and Mrad with / without methylene blue. FIG. 11 is a graph showing the relationship between HGB and Mrad with / without methylene blue. FIG. 12 is a graph showing the relationship between HCT and Mrad with / without methylene blue. FIG. 13 is a graph showing the relationship between MCV and Mrad with / without methylene blue. FIG. 14 is a graph showing the relationship between MCH and Mrad with / without methylene blue. FIG. 15 is a graph showing the relationship between PLT and Mrad with / without methylene blue. FIG. 16 is a graph showing the relationship between MCHC and Mrad with / without methylene blue. FIG. 17 is a graph showing the relationship between RDW and Mrad with / without methylene blue. FIG. 18 is a graph showing the relationship between MPV and Mrad with / without methylene blue. FIG. 19 is a photograph of a titration plate showing antibodies that have preserved biological activity 4 weeks after irradiation according to the present invention. FIG. 20 is a photograph of a titration plate showing light staining of erythrocytes after gamma irradiation according to the present invention and storage at room temperature for 4 weeks. FIG. 21 is a photograph of a portion of a titration plate showing antibodies that have preserved biological activity 4 weeks after irradiation according to the present invention.

Claims (22)

A method of storing functional biological material comprising:
Providing functional biological material;
Irradiating the biological material and preserving its function, and storing the biological material at a first temperature.   Providing a functional biological material includes providing a blood component selected from the group consisting of red blood cells, white blood cells, monocytes, platelets, clotting factors, immunoglobulins, monoglobulins, and polyimmunoglobulins; The method of claim 1.   Preparing functional biological material is cartilage, bone marrow, bone marrow cell suspension, ligament, tendon, nerve, bone, demineralized bone matrix, graft, joint, femur, femoral head 2. The method of claim 1, comprising providing animal tissue selected from the group consisting of: teeth, skin grafts, heart valves, corneas, arteries, veins, organs, carbohydrates and collagen.   Providing the functional biological material comprises providing a non-cytoplasmic material selected from the group consisting of protein, proteinaceous material, enzyme, antigen, amino acid, peptide, sugar, lipid and marrow Item 2. The method according to Item 1.   The method of claim 1, wherein irradiating the biological material comprises providing the biological material with an amount of radiation effective to sterilize the biological material.   The method of claim 1, wherein irradiating the biological material comprises providing the biological material with an amount of gamma radiation effective to sterilize the biological material.   2. The method of claim 1, wherein irradiating the biological material comprises imparting to the biological material an amount of radiation effective to inactivate a virus.   The method of claim 1, wherein irradiating the biological material comprises imparting the biological material with an amount of gamma radiation effective to kill bacteria.   The method of claim 1, wherein irradiating the biological material comprises applying gamma radiation to the biological material with a total exposure of 30 kGy.   Irradiating the biological material may cause the biological material to travel for about 5 minutes. The method of claim 1, comprising applying gamma radiation at a dose of about 0.5 kGy / hour to about 4.0 kGy / minute for a period of time to about 40 hours.   The method of claim 1, wherein storing the biological material comprises storing the biological material at room temperature prior to use.   The method of claim 1, wherein storing the biological material comprises storing the biological material at ambient temperature prior to use.   The method of claim 1, wherein storing the biological material comprises maintaining the biological material at a first temperature of 5 ° C. to 100 ° C.   The method of claim 1, wherein storing the biological material comprises maintaining the biological material at a first temperature of about 10 ° C. to about 30 ° C.   The method of claim 1, further comprising controlling a second temperature of the biological material when irradiating the biological material.   The method of claim 1, further comprising maintaining a second temperature of the biological material between about 10 ° C. and about 30 ° C. when irradiating the biological material. A method for preserving functional biochemical entities,
Preparing functional biochemical entities,
Irradiating said biochemical entity and preserving its function, and storing said biochemical entity at ambient temperature.   18. The irradiation of claim 17, wherein irradiating the biochemical entity includes providing the biochemical entity with an amount of radiation effective to sterilize the biological entity. Method. A method of storing functional biologically active molecules,
Providing biologically active molecules,
Irradiating the biologically active molecule and preserving its function, and storing the biologically active molecule at room temperature.   Irradiating the biologically active molecule includes providing the biologically active molecule with an amount of radiation effective to sterilize the biologically active molecule. The method of claim 19.   A functional biological material produced by the method of any one of claims 1-16. A method of preventing or treating physical condition or disease,
Providing a biological material produced by the method of any one of claims 1-16;
Storing the biological material at ambient temperature and administering an effective amount of the biological material to a patient.

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